Glycosyltransferase mutant, and method for catalytic synthesis of rebaudioside m using glycosyltransferase mutant

Abstract

A glycosyltransferase mutant and a method for catalytic synthesis of rebaudioside M using the glycosyltransferase mutant are provided. The glycosyltransferase mutant is obtained through mutation based on a glycosyltransferase amino acid sequence shown in SEQ ID NO: 1, and can be produced by inducing expression in a mutant strain. The glycosyltransferase mutant can be used as a catalyst for enzymatic catalytic synthesis of 12.8 g/LRebM from 20 g/LRebE. Michaelis constants (a kinetic parameter) of the glycosyltransferase mutant S195Q for rebaudioside E and rebaudioside D are 56.34±2.02 μM and 214.48±14.54 μM, respectively, which are ⅓ and ⅖ of a Michaelis constant of a wild-type (WT) glycosyltransferase, respectively. The glycosyltransferase mutant can be coupled with sucrose synthase (SuSy) to allow the catalytic synthesis of rebaudioside M and a recombinant strain in which a glycosyltransferase UGT76G1 or a mutant thereof is co-expressed with SuSy is constructed to allow the catalytic synthesis of rebaudioside M.

What is claimed is: 
     
         1 . A glycosyltransferase mutant, wherein the glycosyltransferase mutant is obtained through a mutation based on a glycosyltransferase amino acid sequence shown in SEQ ID NO: 1, and the glycosyltransferase mutant is G87D, S147D, S195Q, L200Y, S285W, or G378P. 
     
     
         2 . The glycosyltransferase mutant according to  claim 1 , wherein a nucleotide sequence for the glycosyltransferase mutant G87D is shown in SEQ ID NO: 3, a nucleotide sequence for the glycosyltransferase mutant S147D is shown in SEQ ID NO: 4, a nucleotide sequence for the glycosyltransferase mutant S195Q is shown in SEQ ID NO: 6, a nucleotide sequence for the glycosyltransferase mutant L200Y is shown in SEQ ID NO: 7, a nucleotide sequence for the glycosyltransferase mutant S285S is shown in SEQ ID NO: 9, and a nucleotide sequence for the glycosyltransferase mutant G378P is shown in SEQ ID NO: 10. 
     
     
         3 . An expression gene encoding the glycosyltransferase mutant according to  claim 1 . 
     
     
         4 . A recombinant plasmid carrying the expression gene according to  claim 3 . 
     
     
         5 . A method for synthesizing rebaudioside M using a glycosyltransferase mutant, comprising the following steps:
 1) cloning a coding gene for a glycosyltransferase UGT76G1 mutant and a coding gene for sucrose synthase (SuSy) into an expression vector to obtain a corresponding recombinant plasmid;   2) transforming the recombinant plasmid into  Escherichia coli  ( E. coli ) BL21 (DE3) to obtain a recombinant strain in which the glycosyltransferase mutant and the SuSy are co-expressed;   3) inoculating the recombinant strain into an Luria-Bertani (LB) liquid medium, and adding an inducer to induce expression; centrifuging a resulting bacterial solution at a low temperature, collecting a resulting bacterial precipitate, and resuspending the bacterial precipitate with an appropriate buffer; and subjecting a resulting bacterial suspension to ultrasonication, centrifuging a resulting ultrasonication system, and collecting a resulting supernatant, which is a crude enzyme solution; and   4) adding rebaudioside E or rebaudioside D as a substrate, sucrose, and the crude enzyme solution to a catalytic reaction system, and allowing a reaction at an appropriate temperature; and collecting a sample, inactivating the sample at a high temperature, centrifuging the sample, and collecting a resulting supernatant, which is the rebaudioside M.   
     
     
         6 . The method according to  claim 5 , wherein the inducer is isopropyl-β-D-thiogalactoside, the inducer is added at a concentration of 0.1 mM to 1.0 mM, and the expression is induced for 20 h to 40 h. 
     
     
         7 . The method according to  claim 5 , wherein in the catalytic reaction system, a concentration of the rebaudioside E is 10 g/L to 50 g/L, a concentration of the sucrose is 10 g/L to 200 g/L, and a concentration of a crude enzyme is 1 g/L to 5 g/L; and the reaction is conducted at 20° C. to 50° C. for 2h to 32 h. 
     
     
         8 . The method according to  claim 5 , wherein in the catalytic reaction system, a concentration of the rebaudioside D is 1 g/L to 5 g/L, a concentration of the sucrose is 1 g/L to 20 g/L, and a concentration of a crude enzyme is 1 g/L to 5 g/L; and the reaction is conducted at 20° C. to 50° C. for 2h to 32 h. 
     
     
         9 . The method according to  claim 5 , wherein the SuSy is StSUS1 or McSuSy, a nucleotide sequence for the StSUS1 is shown in SEQ ID NO: 11 and a nucleotide sequence for the McSuSy is shown in SEQ ID NO: 12.